To meet Agency requirement for reliable assessment of botulinum neuron toxin A (BoNT/A) in manufactured products, we propose to apply the fluorescent peptide beacon technology for measurement of both binding function and proteolytic function of the toxin in a single assay, hence "from lab to marketplace". Because this is a homogenous technology, it is amendable to ease of use, portability, automation and high throughput. Here we propose to develop a series of novel fluorescent reagents, to use them in conjunction with immobilized antibodies in a self- contained system to achieve robust and high sensitivity detection of BoNT/A in complex samples. Specifically, our objectives are as follows: Specific Aim #1: Design, synthesis and testing of a novel fluorescent peptide beacon as tracer to detect the binding function of the toxin;Specific Aim #2: Design, synthesis and testing of a novel fluorogenic peptide to detect the proteolytic function of the toxin;Specific Aim #3: Design, synthesis and testing of a novel fusion peptide made of the above two peptides linked together via flexible linker to detect both functions of the toxin. Specific Aim #4: Design and develop a surface immobilization strategy and self-contained sample handling system to increase sensitivity and ease of use. Specific Aim #5: Modify an existing portable fluorescent reader/incubator to use as testbed for measuring fluorescence kinetics, and correlate such measurement with potency units. Based on our experience in working on these types of systems, we feel confident the proposed concepts would be demonstrated feasible. We hope the proposed work would lay a sound foundation to launch this project to the next stage of development, where we would establish correlation of the in vitro testing results with mouse bioassays using complex sample matrices and on developing robust instrumentation. Our long term goal is to seek FDA approval to manufacture and market this method and device for routine deployment in manufacturing plant, regulatory agencies, world health organizations, hospitals and doctor's office, border control, etc. PUBLIC HEALTH RELEVANCE: Botulinum Neuron Toxins (BoNTs) are a class of potent neuron toxins produced by Clostridium botulinum bacterium widely found in nature. While these toxins can cause diseases and casualties in events of accidental infection, food poisoning, or biological attack, they are also widely used to cure diseases and improve the quality of lives for millions of people in over 75 countries. We hereby propose to develop novel reagents and assay system to accurately measure the potency of the toxins in manufactured products to help prevent over- or under- dose. When fully developed, the system could be deployed in manufacturing plant, regulatory agencies, hospitals, and doctor's offices, world health organizations, border control, etc.